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Please be advised that this information was generated on 2021-10-06 and may be subject to change. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20333–20343, June 8, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

X-ray Structures of Progesterone Receptor Ligand Binding Domain in Its Agonist State Reveal Differing Mechanisms for Mixed Profiles of 11␤-Substituted Steroids Received for publication, September 30, 2011, and in revised form, March 3, 2012 Published, JBC Papers in Press, April 25, 2012, DOI 10.1074/jbc.M111.308403 Scott J. Lusher‡1, Hans C. A. Raaijmakers‡, Diep Vu-Pham§, Bert Kazemier§, Rolien Bosch‡, Ross McGuire‡, Rita Azevedo‡, Hans Hamersma‡, Koen Dechering§, Arthur Oubrie‡, Marcel van Duinʈ, and Jacob de Vlieg‡** From the Departments of ‡Molecular Design and Informatics, §Molecular Pharmacology and DMPK, and ʈWomen’s Health, MSD, P. O. Box 20, 5340 BH, Oss, The Netherlands and the **Computational Drug Discovery Group, Radboud University, 6526 HP, Nijmegen, The Netherlands

Background: Understanding the molecular basis for the mixed profiles of progesterone receptor (PR) ligands will benefit Downloaded from future drug design. Results: Two differing mechanisms for the induction of mixed profiles by 11␤-steroids are described. Conclusion: Subtle electrostatic and steric factors explain the differing PR activities of 11␤-steroids. Significance: These observations will impact future drug-design strategies for PR and potentially other nuclear receptors. http://www.jbc.org/ We present here the x-ray structures of the progesterone a fertile area for research, with special focus on the develop- receptor (PR) in complex with two mixed profile PR modulators ment of mixed profile compounds (1–3). Mixed profile modu- whose functional activity results from two differing molecular lators of PR are characterized by decreased transcriptional mechanisms. The structure of Asoprisnil bound to the agonist activity compared with full agonists and increased transcrip- state of PR demonstrates the contribution of the ligand to tional activity compared with full antagonists. Partial agonists, at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 increasing stability of the agonist conformation of helix-12 via a often referred to as selective progesterone receptor modulators 723 specific hydrogen-bond network including Glu . This interac- (SPRMs), have the potential to treat a variety of women’s health tion is absent when the full antagonist, RU486, binds to PR. conditions (4–7) with improved safety and treatment profiles Combined with a previously reported structure of Asoprisnil compared with full agonists or antagonists. bound to the antagonist state of the receptor, this structure Asoprisnil (Fig. 1), demonstrating a mixed agonist/antago- extends our understanding of the complex molecular interac- nist profile depending on tissue type, was the first SPRM to tions underlying the mixed agonist/antagonist profile of the progress to late stage clinical development for the treatment of compound. In addition, we present the structure of PR in its uterine fibroids and endometriosis (8–14). However, the mixed agonist conformation bound to the mixed profile compound profiles of SPRMs are often poorly reflected in classical in vitro Org3H whose reduced antagonistic activity and increased ago- models (15). In our hands Asoprisnil is a full PR antagonist in nistic activity compared with reference antagonists is due to an cell-based transactivation assays (PR agonist EC Ͼ 100 nM,PR induced fit around Trp755, resulting in a decreased steric clash 50 antagonist EC 0.14 nM with 96% efficacy compared with a with Met909 but inducing a new internal clash with Val912 in 50 standard reference (16)) but can be characterized as a SPRM by helix-12. This structure also explains the previously published differences in in vivo models such as the McPhail test (mea- observation that 16␣ attachments to RU486 analogs induce suring endometrial proliferation in immature rabbits) com- mixed profiles by altering the binding of 11␤ substituents. pared with the full antagonist RU486 (17). It has also been Together these structures further our understanding of the shown clinically that Asoprisnil mediates unique endometrial steric and electrostatic factors that contribute to the function of effects in healthy premenopausal women (17). Difficulties in steroid receptor modulators, providing valuable insight for future compound design. characterizing SPRMs in vitro have historically made their identification and characterization problematic, but traditional methods of steroid receptor drug discovery, designed to identify agonists and antagonists, are now being supplemented by Modulation of the progesterone receptor (PR)2 is the mech- new design approaches and assay types, including peptide anism of action for an array of medications and continues to be recruitment and gene expression, better suited to characterizing mixed profile compounds (15). The atomic coordinates and structure factors (codes 4A2J and 4APU) have been The shared domain structure of steroid receptors, such as deposited in the Protein Data Bank, Research Collaboratory for Structural PR, includes a variable N-terminal domain, a highly conserved Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). 1 To whom correspondence should be addressed. E-mail: s.lusher@ DNA binding domain, and a moderately conserved ligand bind- esciencecenter.nl. ing domain (LBD). The LBD combines a number of functions, 2 The abbreviations used are: PR, progesterone receptor; SPRM, selective proges- including hormone binding, receptor dimerization, and bind- terone receptor modulators; LBD, ligand binding domain; r.m.s.d., root mean square deviation; Org3H, 11-pyridinylphenyl steroids (17␤-cyclopro- ing to other co-modulating proteins that play a role in the con- plycarbonyl-16␣-ethenyl-11␤-[4-(3-pyridinyl)phenyl]-estra-4,9-dien-3-one). trol of transcription. Specifically, gene activation requires the

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recruitment of co-modulating proteins to a region of the sur- EC50 0.66 nM with 47% efficacy, PR antagonist EC50 0.61 nM face of the LBD formed by helices 3, 5, and 12. The position of with 38% (28)). helix-12 can be influenced by the nature of the ligand bound to Elucidation of Org3H in complex with PR revealed a second the receptor, allowing ligands to influence the binding of co- mechanism to explain its mixed profile compared with full modulators and consequently gene activation. Our under- antagonists, with Org3H making a reduced clash with Met909 standing of ligand binding to PR has been improved by a num- due to an induced fit around Trp755, which itself now clashes ber of x-ray crystallography studies (18–23) that have also with Val912 in helix-12. As a further consequence, the flipping furthered our understanding of the molecular mechanisms of Trp755 results in formation of an additional new subpocket. underpinning antagonism and partial agonism (16, 17, 24–26). Together these structures improve our molecular understand- In particular, it is well accepted that clashes between some ing of the important steric and electrostatic factors contribut- ligands and Met909 in helix-12 is a major contributing factor to ing to the mixed profile seen for many PR modulators. reduced agonistic activity. One important recent publication describes the x-ray struc- MATERIALS AND METHODS ture of Asoprisnil bound to the antagonist conformation of PR Expression and Purification of PR-LBD—The PR LBD, com- in the presence of the co-repressors NCoR and SMRT (17). The prising residues 678–933, was cloned in pET15b (Novagen). Downloaded from authors report the PR LBD in a conformation divergent from Expression was performed in Escherichia coli BL21(DE3) star the classical agonist state explaining the reduced agonistic (Invitrogen) in 2ϫYT medium by overnight induction at 20 °C activity of the compound compared with full agonists. How- in the presence of 10 ␮M OrgA (Fig. 1). OrgA is a member of a ever, the structure gives only limited explanation for the com- compound class described as glucocorticoid receptor antago-

pound increased agonistic activity compared with other fully nists (28) but is a relatively potent PR partial agonist whose http://www.jbc.org/ antagonistic 11␤-steroids. The x-ray structure of Asoprisnil activity is described in a recent article (26). Bacteria were lysed bound to the antagonistic conformation of PR shows the ligand in buffer A (50 mM Tris, pH 7.8, 250 mM NaCl, 10% glycerol, 10 polar oxime group to be in close contact to hydrophobic resi- mM ␤-mercaptoethanol) with 0.4 mM Pefablock (Roche dues in the two described co-repressors (NCoR and SMRT) Applied Science) and 50 ␮M OrgA and purified on nickel nitri-

(17). RU486 has a less polar dimethyl amine group in the posi- lotriacetic acid. Fractions were eluted with buffer A with 100 at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 tion equivalent to the Asoprisnil oxime, leading the authors to mM imidazole. Elution fractions were collected and treated with hypothesize that RU486 might make stronger hydrophobic 2.5 wt/wt % thrombin (Kordia) overnight at 4 °C to cleave the contacts to co-repressors and, therefore, facilitate their recruit- N-terminal His tag. Thrombin was removed by adding benz- ment, thus explaining the differing biological activity of the two amidine-Sepharose (GE Healthcare), centrifuging for 10 min at compounds. 5000 ϫ g, and harvesting the supernatant. To make the final The same report demonstrates that Asoprisnil bound PR crystallization sample, the protein was dialyzed to buffer A to does recruit co-activators such as SRC1. The ability for the Aso- which 1 mM EDTA, 1 mM dithiothreitol, and 10 ␮M OrgA were prisnil-PR complex to recruit co-activators suggests the com- added and subsequently concentrated in a stirring cell to about plex is able to adopt an agonistic conformation in addition to 4 mg/ml as measured by its absorption at 280 nm. The sample the antagonist conformation seen when in complex with co-re- was stored at Ϫ70 °C in aliquots of 50 ␮l. pressors. This is in line with the equilibrium model for partial Crystallization—Crystals of the PR LBD in complex with agonism (16, 27), which suggests that mixed profile compounds OrgA were grown at room temperature from 3.5-␮l drops are able to partially stabilize their receptors in the agonist con- hanging over a mother liquor of 20–30% polyethylene glycol formation compared with the complete stabilization elicited by 3350, 0.1 M Hepes, pH 6.5, 100 mM Mg2SO4, and 10% (v/v) full agonists. This results in altered patterns of co-modulator glycerol. Drops consisted of 2 ␮l of protein sample and 1 ␮lof recruitment and modified biological outcomes. It also suggests mother liquor and 0.5 ␮l of 40% 1,3-propanediol. Kite-shaped that although the agonistic conformation of PR bound to Aso- crystals usually appeared after about 3 days. prisnil may not be the lowest energy, it remains biologically Ligand Replacement—Crystals of the PR LBD in complex meaningful. with OrgA were collected and transferred to mother liquor to In an attempt to identify the basis for the ligands retained which 0.25 mM concentrations of either Asoprisnil or Org3H agonistic activity compared with full antagonists such as RU486 were added. 0.25 mM is a significant excess of either compound. (Fig. 1), we have used a previously described soaking technique The crystals were stored in a sitting drop at room temperature. (16, 26) to determine the x-ray structure of the PR-Asoprisnil The solutions surrounding the crystals were replaced by fresh complex in its agonist state. This has allowed us to identify an solution 10 times over a period of 2 weeks. After this period, the interaction between the 11␤-benzaldoxime of Asoprisnil and crystals were frozen and transferred for data collection. Glu723 that helps partially stabilize helix-12 in its agonist Data Collection—Diffraction data were collected using a conformation. Rigaku rotating-anode x-ray generator operating at 100 mA To establish if this mechanism is universal for 11␤-substi- and 50 kV. The dataset for the PR-Asoprisnil complex was col- tuted steroids, we have also solved the x-ray structure of an lected to 2.08 Å at 100 K and processed with mosflm/scala, and in-house SPRM from a class of 11-pyridinylphenyl steroids the data for the PR-Org3H complex were collected to 1.9 Å at (17␤-cycloproplycarbonyl-16␣-ethenyl-11␤-[4-(3-pyridinyl)- 100 K and processed with d*TREK/scala (29). Structures were phenyl]-estra-4,9-dien-3-one) that we refer to as Org3H (Fig. solved and refined using the CCP4i interface of the CCP4 soft- 1), with a previously disclosed mixed PR profile (PR agonist ware suite (30). Data are summarized in Table 1. Although of

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TABLE 1 available x-ray studies and generate new x-ray structures to Final crystallographic data and refinement statistics address this question. Data in parentheses indicate the last resolution shell. Equilibrium Model for Steroid Receptor Function Suggests Structure PR-Asoprisnil PR-Org3H Both Agonistic and Antagonistic Conformations Are Relevant Space group P21 P21 for Mixed Profile Compounds—It has been known since the Unit cell (Å3) 58.61 64.70 69.99 57.66 64.37 70.47 ␤-Angle (°) 95.7 96.3 early 1990s that agonists and antagonists induce different con- Resolution (Å) 43.3-2.08 (2.19-2.08) 47.4-1.90 (2.00-1.90) formational changes in PR (35, 36) with the demonstration that Completeness (%) 99.8 (100) 100 (99.9) Rmerge 0.103 (0.496) 0.082 (0.729) the C-terminal is able to adopt two different positions corre- Mn2ϩ (I/␴) 6.3 (2.0) 8.7 (2.1) sponding to the biological activity of the ligand (35, 37). Both of Multiplicity 3.1 (3.0) 4.1 (4.0) R factor/Rfreeb 20.4/25.1 20.8/24.5 these conformations are distinct from the conformation of the Atoms 4217 4289 unliganded. It was later shown that a third conformation could Protein atoms 3967 4047 ␣ Ligand atoms 66 64 be detected in the presence of 16 -substituted analogs of Water molecules 78 173 RU486 (38) known to be mixed-profile PR modulators. It has Other molecules e.g. sulfate 5 5 r.m.s.d. bonds, Å 0.011 0.078 since been postulated that full agonists promote interaction r.m.s.d. angles (°) 1.2 1.05 with co-activators, and full antagonists change the conforma- Downloaded from B-factors (average Å2) Main chain 36.0 40.5 tion of the receptor to inhibit co-activator binding in favor of Side chain 38.2 42.0 co-repressor binding (10). In relation to this, mixed-profile Water molecules 22.5 30.8 compounds induce an intermediate state of interaction Ligand A chain 30.9 50.1 (OrgA) Ligand B chain 29.3 37.5 (Org3H) between receptor and co-modulators (38–40). The equilib- 2 Wilson B-factor (Å ) 29.3 38.5 rium model for nuclear receptor agonism/antagonism suggests http://www.jbc.org/ PDB identifier 4A2J 4APU that mixed profile ligands partly stabilize the agonist conformation of their receptor (16, 27) and allow co-activators to bind similar resolution, the Asoprisnil dataset is somewhat weaker but with less efficacy than full agonists. The ability for the Aso- than the Org3H dataset, and less water molecules were used prisnil-PR complex to recruit co-activators (17) suggests the

describe the electron density. complex is able to adopt an agonistic conformation in addition at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 Modeling and Visualization—All figures have been gener- to the antagonist conformation seen when in complex with co- ated using PyMOL (The PyMOL molecular graphics system, repressors. It has also been shown for the steroid receptors that Schrodinger, LLC). Ligand structures were optimized using the the presence or absence of co-modulating proteins can alter the semi-empirical quantum mechanical MOPAC module in specific conformation of structures resulting from x-ray crys- YASARA (31). YASARA was also used to calculate ligand vol- tallography even after co-expression studies. For example, the umes, with all figures representing the volume of the solvent x-ray structure of genistein bound to the estrogen receptor-␤ accessible surface. has been reported in an agonist conformation in the presence of co-activator but also in a conformation similar to the classic RESULTS AND DISCUSSION antagonist conformation in the absence of co-activator (41, 42). Size of 11␤ Group Does Not Correlate to Antagonistic Activity— The binding of co-modulating proteins is itself a driving force in The generally accepted mechanism for introducing PR antago- the equilibrium and not just a consequence. The relative bal- nism into steroidal and non-steroidal compounds is via the ance of co-activator and co-repressor expression within a given attachment of a bulky group at the steroids 11␤ position or an target cell determines the relative agonist versus antagonist equivalent position in non-steroidal compounds. This group activity of mixed-profile compounds (40). Although the previ- should clash with helix-12 and preclude it from adopting its ously reported structure of PR bound to Asoprisnil (from co- agonistic conformation. Impeding helix-12 from adopting its expression studies) shows the receptor in an antagonist confor- agonistic position prevents the correct formation of the AF-2 mation, it is in the presence of co-repressor. The ability of the surface and thus excludes the binding of co-activators. This PR-Asoprisnil complex to recruit co-activators indicates the mechanism is supported by the x-ray structures of PR bound to agonist conformation of the receptor bound to Asoprisnil is RU486 (16) and Asoprisnil (17) and structures of related recep- both viable and biologically meaningful. This conclusion led us tors such as the AR and glucocorticoid receptor (32–34). How- to utilize our previously described PR soaking method (16, 26) ever, it has become clear to us that not all bulky groups are equal to study Asoprisnil bound to a fixed agonistic conformation of in this respect and that more subtle factors must determine how PR to determine whether this structure would give additional successfully each substituent is able to preclude helix-12. For insight into the molecular basis for Asoprisnil retained agonism example, the side chain of the antagonist RU486 has a volume of compared with RU486. 436 Å3, which although larger than the side chain of the SPRM Asoprisnil Successfully Replaces OrgA during Soaking Ex- Asoprisnil at 385 Å3, is somewhat smaller than the side chain of periments—OrgA is a nonsteroidal compound (Fig. 1) with a the second SPRM Org3H at 504 Å3. chemical structure distinct from 11␤-steroids whose PR activ- This inconsistency suggests that steric contributions alone ity and binding is well described in an earlier publication to do not guarantee the generation of full antagonists. It also sug- which we refer readers (26). PR forms homodimers endoge- gests that other factors must be involved in the inducement of nously and also after crystallization. Fig. 2 shows the electron mixed profiles and that understanding these factors may help in density around Asoprisnil from the B monomer of a new x-ray the future design of SPRMs. This promoted us to review the structure of PR in its agonistic conformation. The clarity of the

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electron density around the ligand allows us to conclude with a tion compared with the poorly ordered helix-12 as observed high degree of confidence that OrgA has been completely when bound to RU486. This is consistent with Asoprisnil replaced by Asoprisnil during the soaking. The exchange is reduced antagonistic activity and its ability to recruit co-activa- complete for both monomers with the ligand in an almost iden- tors. There is, however, disorder in region 895–905, the con- tical binding mode. To aid in comparison with the previously necting loop between helix-11 and helix-12 of both monomers published RU486 structure in which the ligand soaks into only in the crystal. The loops of both monomers touch through a the B monomer, we will describe only the equivalent monomer symmetry contact and are also disordered in both PR-antago- from the new PR-Asoprisnil complex. nist structures bound to Asoprisnil. Helix-12 Is Well Ordered in Its Agonist Conformation—This Soaking Asoprisnil into PR-Agonist Crystals Results in Same PR Asoprisnil complex is quite different from the public PR- Ligand Binding Mode as Co-expression—Despite the new PR- Asoprisnil structures (PDB access codes 2OVH and 2OVM) Asoprisnil structure being in an agonistic conformation rather and from the PR-RU486 structure (PDB access code 2W8Y). than the antagonistic conformation of the previously reported The difference between the previously published PR-Asoprisnil PR-Asoprisnil structures (17), the binding mode of the ligand is structures and this new structure is mainly located between

comparable. Asoprisnil orients itself almost identically within Downloaded from residues 881 and 924, as the classic antagonistic displacement of helix-12 does not occur in this new structure (Fig. 3). Exclud- the agonist and antagonist conformations of PR, making the ing the C-terminal residues after helix-11, the PR-agonist and same contacts with the receptor. The 3-keto group makes the 725 766 PR-antagonist structures bound to Asoprisnil show a root classic interactions to Gln and Arg typical for oxosteroids, ␣ mean square deviation (r.m.s.d.), calculated for their C␣ atoms, with the 17 attachment occupying a hydrophobic pocket con- 715 718 794 797 801 890 of 0.65 Å. Helix-12 is well ordered and in the agonist conforma- sisting of Leu , Leu , Phe , Leu , Met , and Tyr http://www.jbc.org/ (Fig. 4). This pocket, referred to as the 17␣ pocket, has previously been described for a structure of PR bound to mometasone furoate (21) and appears to provide additional room for ligand expansion irrespective of agonism or antagonism.

No direct or indirect polar interaction exists between the at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 ligand and Asn719. The presence of a water-bridged interaction between steroidal ligands and Asn719 has been described in other PR-ligand complexes, including the binding of RU486 and norethindrone (26), but the 17␤-methoxy group of Aso- prisnil sterically precludes the presence of a water molecule at this position. As can be seen in Fig. 5, the steroid scaffolds of Asoprisnil from both the PR-agonist and PR-antagonist complexes are relatively well overlaid with a small shift downward seen in Aso- prisnil bound to the agonist conformation of PR. The 17␣ and 17␤ attachments of both compounds are also well overlaid. The most significant difference between Asoprisnil bound to the agonist conformation and the antagonist conformation of PR is a 0.9 Å adjustment in the position of the oxime group. In the agonistic complex the oxime and steroid core appear to sit lower to reduce the clash with Met909 in helix-12. Because of the displacement of helix-12 in the antagonistic complex, this FIGURE 1. The structures of progesterone (A), norethindrone (B), RU486 clash is not possible. (C), Asoprisnil (D), Org3H (E), and OrgA (F) are shown.

␦ ؊ 909 FIGURE 2. 2Fo DFc OMIT electron density maps around the ligand and Met for RU486 (A), Asoprisnil (B), and Org3H (C) are shown at 1. 0 . Electron densities suggest a more stable conformation of Met909 in the Asoprisnil and Org3H structures compared with the RU486 complex.

20336 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 24•JUNE 8, 2012 Differing Mechanisms Explain Mixed Agonist/Antagonist Profile of 11␤-Steroids Downloaded from FIGURE 3. Panel A shows the secondary structure of the PR-Asoprisnil complex generated by a soaking experiment described in this article. Helix-12 is colored red and is oriented in the classic agonist conformation. Panel FIGURE 5. Asoprisnil from the PR-agonist conformation is shown (car- B shows the secondary structure of a previously described PR-Asoprisnil bons are colored green) and overlaid with Asoprisnil from the PR-antag- complex generated (PDB code 2OVH) by co-expression with the ligand. onist conformation (carbons are colored magenta). Asoprisnil from the Helix-12 is colored red and is shifted from the agonist position to allow the PR-agonist conformation sits slightly lower in the pocket compared with Aso- binding of a co-repressor peptide, colored blue. Protein-ligand complexes prisnil from the PR-antagonist conformation. http://www.jbc.org/ were aligned using the Motif function in YASARA (31), and images were generated using PyMOL. at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018

FIGURE 6. Overlaid structures of PR-agonist structure bound to RU486 (carbons are colored orange) compared with PR-agonist structure bound FIGURE 4. Binding mode of Asoprisnil with hydrogen bonding from to Asoprisnil (carbons are colored green). Hydrogen bonds between the Gln725 766 and Arg to the ligands 3-keto group, highlighted in magenta. ligand keto groups to Gln725 and Arg766 are shown in magenta. Helix-12 is Residues forming a hydrophobic pocket surrounding the 17␣ group are also colored red with Met909 highlighted in both complexes (carbons are colored shown. red). The ligands overlay closely and appear to sterically impact on Met909 to a similar degree. Met909 Is Better Ordered in Asoprisnil Complex Compared with Equivalent RU486 Structure—In the past our group pub- each complex shown in Fig. 2. The increased order of helix-12 lished an x-ray structure of RU486 bound to the agonist con- and particularly Met909 in the Asoprisnil structure is consistent formation of PR (16), which clearly demonstrated disorder of with the increased agonistic activity of the ligand compared Met909 in helix-12 due to a clash with the ligand (Fig. 2A). This with RU486. clash resulted in the destabilization of helix-12, measured by As we investigated the molecular basis for this difference, we an increase in the b-factors of the helix compared with considered the possibility that the reduced volume of Asopris- helix-12 bound to previously described full agonists. This nil oxime and its linear rather than branched nature, compared provided a compelling model to explain the antagonistic with the dimethylamine of RU486, may reduce its clash with activity of RU486. Met909. However, comparison of the overlaid ligands did not Met909 in the PR-agonist complex bound to Asoprisnil does suggest a significant difference in their spatial arrangements adopt a modified conformation compared with its position around the position of Met909 (Fig. 6), and therefore, other fac- bound to other full agonists, but the new rotamer is well tors must be responsible that will be discussed in the following ordered, as can be seen when comparing the electron density of section.

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723 908 909

FIGURE 7. Panel A illustrates the stabilizing interaction between Glu and helix-12 (colored red) by hydrogen bonding to the backbone of Met and Met . Downloaded from Panel B shows the binding mode of Asoprisnil bound to the agonist conformation of PR with ligand to receptor hydrogen bonds shown in magenta. Interactions between Glu723 and helix-12 (shown in red) are also shown. Panel C shows a comparison between the effect of binding Asoprisnil (green)toPRand raloxifene (blue) to estrogen receptor ␣. Binding of Asoprisnil to PR contributes the stabilizing interactions between Glu723 and the backbone of helix-12. On the other hand, the binding of raloxifene to estrogen receptor ␣ results in Asp351 losing its interactions with the backbone of helix-12 as it now prefers to re-orientate and make a salt bridge to the ligands basic amine.

Asoprisnil Oxime Group Contributes to Stabilization of its usual function of stabilizing the agonistic conformation of http://www.jbc.org/ Helix-12 in Its Agonistic Conformation—As previously de- helix-12 (27, 41, 44–51, 53). scribed (16, 43), Glu723 in helix-3 plays an important role in SERMs are, therefore, well characterized examples of ligands stabilizing the agonist conformation of helix-12 by hydrogen whose mixed profiles can partly be attributed to disrupting the bonding to main chain amines in both Met908 and Met909 (Fig. stabilizing interaction of the conserved acidic residue at this 723

7A). In addition, Glu is also able to make a hydrogen bond to position. We believe that Asoprisnil is the first characterized at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 a water molecule in the bulk solvent. In the antagonistic con- example of a ligand whose mixed profiles can be attributed to 723 formation of PR bound to Asoprisnil, Glu points away from strengthening this same stabilizing interaction. the ligand to make polar contacts with the solvent and, due to Interaction with Glu723 Is Unlikely to Explain Partial Ago- the displacement of helix-12, no longer makes stabilizing inter- nism of Org3H—The interaction between Asoprisnil and Glu723 908 909 actions to Met and Met . is dependent on the presence of the 11␤-benzaldooxime group In the agonistic conformation of PR-bound Asoprisnil, and as such is unlikely to be a universal model for mixed profile 723 Glu , as expected, stabilizes the agonist position of helix-12 compounds. For example, Org3H is a SPRM from a class of but, additionally, makes a hydrogen bond to the ligand oxime ␤ 723 steroids incorporating 11 -pyridinylphenyl groups and, there- group (Fig. 7B). In this new state Glu strengthens the hydro- fore, lacks the hydrogen-bond donating capacity of the Asopris- gen-bond network that stabilizes helix-12 in its agonistic posi- nil oxime substituent. In an attempt to explain its partial ago- tion. This interaction is not duplicated on binding of RU486, nism, we selected Org3H as a candidate for characterization via which suggests the agonist conformation of PR bound to Aso- x-ray crystallography following the same soaking procedure as prisnil is more stable than the agonist conformation of PR we have described for Asoprisnil. bound to RU486. This relative increased stability of the agonist Org3H Is Bound to Monomer B but Not to Monomer A—As is conformation when bound to Asoprisnil explains the ability of the case for our PR-Asoprisnil structure, the asymmetric unit this complex to recruit co-activators and suggests that the contains two copies of the PR-LBD. Visual inspection of the existence of the interaction with Glu723 pushes the receptor equilibrium toward agonism compared with RU486. electron density within the ligand binding pocket of the struc- Glu723 Is Equivalent to Asp351, Residue Crucial for Agonist/ ture clearly shows that monomer B contains Org3H but OrgA is Antagonist Balance in Estrogen Receptor ␣—Glu723 in PR is still present in monomer A and has not been replaced during equivalent, based on sequence and structure alignments, to the soaking. A similar observation was made when RU486 Asp351 in the estrogen receptor ␣, which plays a similar role in soaked into the B monomer of PR-norethindrone crystals stabilizing the agonist conformation of helix-12. The impor- but failed to displace the original ligand from monomer A tance of this residue has been demonstrated by studying the (16). As Asoprisnil displaced OrgA in both monomers, we naturally occurring D351Y mutation and other synthetic muta- can conclude that monomer A is accessible in the crystal but tions that result in loss of receptor agonistic activity, consistent must have lower affinity for Org3H or a slower rate of ligand with the residues normal role in stabilizing the agonist confor- entry compared with monomer B. As hypothesized in the mation of the receptor. Selective modulators of estrogen recep- RU486 study, this difference is likely to arise from conforma- tor ␣, referred to as SERMs and including raloxifene and tional differences in the two crystallographically independ- tamoxifen, contain an important basic amine function that is ent PR LBDs in the crystals. These differences are most almost ubiquitous among this drug class. The role of this nitro- prominent around the loop 785–808, a region previously gen is to form a salt bridge to Asp351, requiring the amino acid hypothesized to be a route of entry for the ligand during the to adopt a new conformation and prevent it from undertaking soaking experiments (16).

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FIGURE 8. Panels A and B show the overlaid structures of Asoprisnil (carbons are in green), RU486 (carbons are in orange), and Org3H (carbons are in cyan). The secondary structure of the PR-Asoprisnil structure is shown as a schematic representation in green. The 17␣ side chains of Asoprisnil and

RU486 overlay relatively closely with the 16␣ substituent of Org3H due to the Downloaded from displacement of this compounds D-ring. In A, positions 16 and 17 of Org3H and Asoprisnil are labeled. In B, position 16 of Org3H and Asoprisnil was labeled.

FIGURE 9. Overlaid structure of Asoprisnil (carbons are in green), RU486 Steroidal Core of Org3H Binds Differently Than Other (carbons are in orange), and Org3H (carbons are in cyan). The secondary

Steroids—The binding of OrgA in monomer A is practically structure of the PR-Asoprisnil structure is shown as a schematic representa- http://www.jbc.org/ tion in green. The 11␤ substituents of Asoprisnil and RU486 overlay closely, identical to the previously described PR structures containing but the 11␤ substituent of Org3H is clearly divergent. OrgA and requires no further description (26). Org3H shares the classical interaction with Gln725 and Arg766 via its 3-keto group, with the A-ring in general binding in the conventional

manner described for RU486 and Asoprisnil. In the second at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 monomer Org3H shares the same scaffold as RU486 and Aso- prisnil, but does not overlay as closely as these two ligands after binding to the receptor (Fig. 8). Although the conformation of the ligand is unchanged, the Org3H binding mode shows distinct unexpected differences, mostly around the D-ring, compared with the other steroids as it is shifted away from its typical position toward the 17␣ pocket. Org3H does not have a 17␣ attachment, as seen for RU486 and Asoprisnil, but the reposi- 755 tioning of the D-ring is significant enough that the vinyl group FIGURE 10. Panel A shows Trp from the Asoprisnil-bound complex (carbons are in green) and from the RU486-bound complex (carbons are in on position 16␣ of Org3H is oriented in a similar position to the orange) showing the normally observed rotamer for this residue. In con- 17␣ groups of RU486 and Asoprisnil, occupying a comparable trast, Trp755 from the Org3H bound complex is shown (carbons are in cyan), demonstrating its ligand induced movement. Panel B shows a similar scene to space within the receptor as shown in Fig. 8, A and B. This panel A but with Org3H superimposed to show the clash it would make to overlay would not be possible if the steroid scaffold bound in Trp755 if the amino acid was not shifted. the classic position. Induced Fit around Trp755 Relieves Clash with Met909—The The pyridinylphenyl side chain is able to occupy a channel scaffold carbon atoms at C11 in RU486, Asoprisnil and Org3H pointing toward His888 to which it makes a water-bridged are all located at approximately the same position in their var- H-bond from the ligand pyridine nitrogen shown in Fig. 12. In ious co-crystals. The pendant attachments of RU486 and Aso- addition to His888 and the ligand, the conserved water is also prisnil, attached to C11, are also well overlaid and conserved. able to hydrogen-bond to the backbones of Tyr753 and Met756. The phenyl group attached at C11 of Org3H is, however, signif- It has previously been observed that the Trp741 flip in AR icantly divergent from the other reference compounds (Fig. 9). (equivalent to Trp755 in PR) makes available a channel and The pyridinylphenyl fills a pocket resulting from a rearrange- potential interaction with His874 (equivalent to His888 in PR), ment of Trp755. including the presence of the conserved water molecule. Com- Upon binding of Org3H, Trp755 rotates 120° around the bond parison of all publicly available PR structures shows that this between the residues C␣ and C␤ bond and 100° around the channel has not previously been described for this receptor (Fig bond between C␤ and C␥ (shown in Fig. 10, A and B). The new 13). It appears that the unusual orientation of Org3H steroidal orientation of Trp755 now packs against Leu726 in helix-3, pro- core is required to allow the 11␤ side chain to adopt this viding space to accommodate the side chain of Org3H (shown position. in Fig. 11, A and B). The capacity for Trp755 to adopt novel Effect of 16␣ Attachment Explains Structure Activity Rela- positions has been described when the receptor binds to non- tionship, Suggesting That This Substituent Induces Partial steroidal compounds (24–26). In addition to these examples, Agonism—McDonnell and co-workers (38) have previously the same plasticity has been noted for the equivalent residue, reported the observation that 16␣ attachments on RU486 ana- Trp741, in AR (34, 54–59). logs result in partial agonist activity rather than the full antag-

JUNE 8, 2012•VOLUME 287•NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 20339 Differing Mechanisms Explain Mixed Agonist/Antagonist Profile of 11␤-Steroids

FIGURE 11. Panel A shows a stick representation of Trp755 and a surface representation of the binding pocket of the PR-agonist structure bound to Aso- prisnil (ligand hidden for clarity). Panel B shows a stick representation of 755

Trp and a surface representation of the binding pocket of the PR-agonist Downloaded from structure bound to Org3H (the ligand is hidden for clarity). The flipping of FIGURE 13. The available PR LBD structures in the PDB were aligned using Trp755 in the Org3H-bound complex expands the overall size of the pocket the MOTIF function in YASARA and the superimposed ligands extracted. and provides the opportunity for novel structure-based drug design. A molecular surface encompassing the sum of the ligands was calculated by PyMOL and shown is cyan. Org3H was added to the ligand alignment, and its molecular surface was calculated and displayed as a red mesh. The portion of the red mesh visible represents the additional space required for the toler- ance of this ligand that has not previously been described for this receptor. http://www.jbc.org/ Met909 compared with RU486, which is in agreement with the increased agonistic activity of Org3H compared with RU486. Met909 adopts a different conformation compared with the rotamer reported when binding to the full agonist norethin-

drone (16, 21) but appears to be well tolerated (Fig. 2), even at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 making favorable hydrophobic contacts to the phenyl in the ligands 11␤ attachment. This prompted us to hypothesize that the remaining clash with Met909 may not be significant enough on its own to explain the reduced agonistic activity Org3H compared with full agonists. We, therefore, continued examining the structure to identify if any other factors were at play. In its new position the Trp755 side chain is directed toward Val912 in helix-12 and would clash with the position of this residue in either the RU486 or Asoprisnil (ago conformation) structures, as they would be within 2 Å. To relieve this potential clash, Val912 is pushed away (ϳ1.5 Å average r.m.s.d. across all non-hydrogen atoms but as much as ϳ2.5 Å for some of the side-chain atoms), and we observed a shift of helix-12 not present in agonist structures or either the PR-agonist-Asoprisnil or FIGURE 12. Binding mode of Org3H with the water (oxygen is shown as a PR-agonist-RU486 structures (Fig 14). We previously described red sphere)-mediated hydrogen-bond to His888 is highlighted. Additional a PR structure bound to a full antagonist, OrgB, that also 756 interactions between the water molecule and Met are also shown. induces a flipping of Trp755 (26) but does not result in the clash with Val912 or the displacement of helix-12. So far, the indirect onism that would otherwise be expected. They also suggest that destabilization of helix-12 independent of Met909 appears to be the basis for this effect is by altering the manner in which the unique to this series of compounds and may explain why these crucial 11␤ group of RU486 and its analogs interact with the compounds are clearly mixed profile as measured in cell-based receptor but that crystallographic data would be needed to con- in vitro assays, which typically characterize SPRMs as full firm this. Our crystallography studies show that due to the 16␣ antagonists. attachment on Org3H, its steroidal scaffold binds in an atypical Stabilized Receptor Conformations Provide Valuable Ap- fashion that consequently leads to the ligands 11␤ having a proach for Studying Mixed Profile Modulators—Understanding reduced clash with Met909. Our PR-Org3H x-ray structure, the the molecular basis for mixed profile compounds is hampered first of PR bound to a 16␣-substituted PR modulator, supports by the difficulty in determining relevant co-crystal structures. McDonnell’s hypothesis and suggests that the changing inter- Full agonists stabilize the receptor and, specifically, helix-12 in action between the receptor and the 11␤ groups they observed a conformation suited to binding co-activating proteins, and is due to the utilization of the newly formed pocket behind full antagonists stabilize the receptor in a conformation suited Trp755. to binding co-repressing proteins. The apparent reason for the New Position of Trp755 Clashes with Helix-12—The divergent difficulty in co-crystallizing mixed profile compounds is that position of the Org3H side chain results in a reduced clash with they do not fully stabilize the receptor in either conformation,

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first indication that SPRMs can illicit their responses without directly impeding helix-12 or clashing with Met909. A combination of the ligand-induced clash between Trp755 and Val912 in helix-12 and the limited clash with Met909 suggest a unique mechanism for the Org3H mixed profile. Overall we suggest specific new directions for the design of SPRMs including the exploration of the pocket behind Trp755, modification of interactions with Glu723, and the generation of indirect clashes with helix-12. Conclusion—We present here two new PR structures bound to partial agonists. The structures demonstrate that two distinct mechanisms explain the mixed profiles of the two ligands, indicating that both steric and electrostatic factors can contribute to this mixed PR activity. The additional information that Downloaded from FIGURE 14. Trp755 and helix-12 from Asoprisnil-bound complex (green) has been learned from the binding of Asoprisnil bound to the with Trp755 and helix-12 from RU486-bound complex (orange) com- agonist conformation of PR illustrates the value of studying 755 pared with Trp and helix-12 from the Org3H-bound complex (cyan). mixed profile compounds bound to both the agonist and antag- The alternate position of Trp755 from the Org3H complex would clash with Val912 from the Asoprisnil and RU486 complexes. This clash is relieved by a onist conformations of their receptors as a useful tool for drug rotation of Val912 and a pushing away of the backbone of helix-12 in this design. This also supports the growing use of stabilized recep- region, suggesting a destabilization of the agonist position of helix-12. tor systems either by mutation or following soaking strategies http://www.jbc.org/ as shown here to study otherwise inaccessible receptor-ligand adopting some degree of equilibrium between the two. This complexes. equilibrium allows mixed profile compounds to bind unique patterns of co-modulators compared with full agonists and Acknowledgments—We express our gratitude to Joost Uitdehaag and

antagonists, resulting in their potentially interesting biological Judith Versteegh for earlier contributions to the soaking strategy for at RADBOUD UNIVERSITEIT NIJMEGEN on April 17, 2018 effects. Unfortunately, as a result it also renders them poorly the progesterone receptor and additionally thank Jos Rewinkel and his suited to co-crystallization studies (60). Recently we have seen team for their design and synthesis efforts. the first publications describing methods to circumvent this problem either by introducing stabilizing mutations into the receptor (52, 56, 60) or by generating stable crystals of the REFERENCES receptor using a receptor stabilizing ligand and then exchang- 1. Spitz, I. M. (2003) Progesterone antagonists and progesterone receptor ing this compound with other compounds of interest via soak- modulators. An overview. Steroids 68, 981–993 2. Madauss, K. P., Stewart, E. L., and Williams, S. P. (2007) The evolution of ing (16). Both approaches have the potential to dramatically progesterone receptor ligands. Med. Res. Rev. 27, 374–400 increase our understanding of the biological mechanisms 3. Winneker, R. C., Fensome, A., Zhang, P., Yudt, M. R., McComas, C. C., and underpinning partial agonism and provide novel insight for Unwalla, R. J. (2008) A new generation of progesterone receptor modula- drug optimization. tors. Steroids 73, 689–701 Implications of New PR Structures for Drug Design—All 4. Spitz, I. M., and Chwalisz, K. (2000) Progesterone receptor modulators ligands elicit their behaviors by a combination of their steric and progesterone antagonists in women’s health. Steroids 65, 807–815 5. Benagiano, G., Bastianelli, C., and Farris, M. (2008) Selective progesterone and electrostatic character. The binding of PR agonists is the receptor modulators 3. Use in oncology, endocrinology, and psychiatry. result of a well described combination of steric complementa- Expert. Opin. Pharmacother. 9, 2487–2496 rity and specific electrostatic interactions, typically to Asn719, 6. Benagiano, G., Bastianelli, C., and Farris, M. (2008) Selective progesterone Gln725, and Arg766. Our understanding of the additional inter- receptor modulators 2. Use in reproductive medicine. Expert. Opin. Phar- actions that differentiate full antagonists and SPRMs from full macother. 9, 2473–2485 7. Benagiano, G., Bastianelli, C., and Farris, M. (2008) Selective progesterone agonists have until now been limited to the steric properties of ␤ receptor modulators 1. Use during pregnancy. Expert. Opin. Pharmaco- bulky 11 substituents. The binding mode of Asoprisnil to the ther. 9, 2459–2472 agonist state of PR is the first description of how additional 8. Chen, W., Ohara, N., Wang, J., Xu, Q., Liu, J., Morikawa, A., Sasaki, H., electrostatic factors, specifically the interaction with Glu723, Yoshida, S., Demanno, D. A., Chwalisz, K., and Maruo, T. (2006) A novel can alter biological properties of SPRMs. Altering the nature of selective progesterone receptor modulator asoprisnil (J867) inhibits pro- that interaction by compound optimization has the potential to liferation and induces apoptosis in cultured human uterine leiomyoma cells in the absence of comparable effects on myometrial cells. J. Clin. fine-tune the characteristics of SPRMs with the aim to improve Endocrinol. Metab. 91, 1296–1304 their therapeutic response. 9. Chwalisz, K., Larsen, L., Mattia-Goldberg, C., Edmonds, A., Elger, W., and In addition to this new insight into the potential value of Winkel, C. A. (2007) A randomized, controlled trial of asoprisnil, a novel modifying SPRMs via an electrostatic approach, the PR-Org3H selective progesterone receptor modulator, in women with uterine leio- co-crystal also provides valuable new information regarding the myomata. Fertil. Steril. 87, 1399–1412 steric influences that will also benefit future drug design. In 10. Chwalisz, K., Perez, M. C., Demanno, D., Winkel, C., Schubert, G., and ␤ Elger, W. (2005) Selective progesterone receptor modulator development particular we show how modifications away from the 11 and use in the treatment of leiomyomata and endometriosis. Endocr. Rev. group, such as the presence of 16␣ groups, can influence the 26, 423–438 effect of the classic bulky substituent. This complex is also the 11. DeManno, D., Elger, W., Garg, R., Lee, R., Schneider, B., Hess-Stumpp, H.,

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JUNE 8, 2012•VOLUME 287•NUMBER 24 JOURNAL OF BIOLOGICAL CHEMISTRY 20343 X-ray Structures of Progesterone Receptor Ligand Binding Domain in Its Agonist State Reveal Differing Mechanisms for Mixed Profiles of 11 β-Substituted Steroids Scott J. Lusher, Hans C. A. Raaijmakers, Diep Vu-Pham, Bert Kazemier, Rolien Bosch, Ross McGuire, Rita Azevedo, Hans Hamersma, Koen Dechering, Arthur Oubrie, Marcel van Duin and Jacob de Vlieg J. Biol. Chem. 2012, 287:20333-20343. doi: 10.1074/jbc.M111.308403 originally published online April 25, 2012 Downloaded from Access the most updated version of this article at doi: 10.1074/jbc.M111.308403

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